Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/041—Heterocyclic compounds
  • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
  • A61K51/0455—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
  • C07B59/002—Heterocyclic compounds
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances

Definitions

• the present invention relates to novel, selective, radiolabelled mGluR2 ligands which are useful for imaging and quantifying the metabotropic glutamate receptor mGluR2 in tissues, using positron-emission tomography (PET).
• PET positron-emission tomography
• the invention is also directed to compositions comprising such compounds, to processes for preparing such compounds and compositions, to the use of such compounds and compositions for imaging a tissue, cells or a host, in vitro or in vivo and to precursors of said compounds.
• Glutamate is the major amino acid neurotransmitter in the mammalian central nervous system. Glutamate plays a major role in numerous physiological functions, such as learning and memory but also sensory perception, development of synaptic plasticity, motor control, respiration, and regulation of cardiovascular function. Furthermore, glutamate is at the centre of several different neurological and psychiatric diseases, where there is an imbalance in glutamatergic neurotransmission.
• Glutamate mediates synaptic neurotransmission through the activation of ionotropic glutamate receptor channels (iGluRs), and the NMD A, AMPA and kainate receptors which are responsible for fast excitatory transmission.
• iGluRs ionotropic glutamate receptor channels
• glutamate activates metabotropic glutamate receptors (mGluRs) which have a more modulatory role that contributes to the fine-tuning of synaptic efficacy.
• mGluRs metabotropic glutamate receptors
• Glutamate activates the mGluRs through binding to the large extracellular
• mGluRl-8 Eight different subtypes of mGluRs have been identified (mGluRl-8) which can be divided into three groups based on sequence homology, transduction mechanism and agonist pharmacology. The mGluR2 subtype is negatively coupled to adenylate cyclase via activation of
• Gai-protein Gai-protein, and its activation leads to inhibition of glutamate release in the synapse.
• mGluR2 receptors are abundant mainly throughout cortex, thalamic regions, accessory olfactory bulb, hippocampus, amygdala, caudate-putamen and nucleus accumbens.
• Activating mGluR2 was shown in clinical trials to be efficacious to treat anxiety disorders.
• activating mGluR2 in various animal models was shown to be efficacious, thus representing a potential novel therapeutic approach for the treatment of schizophrenia, anxiety, depression, epilepsy, drug addiction/dependence,
• Parkinson's disease Pain, sleep disorders and Huntington's disease.
• a new avenue for developing selective compounds acting at mGluRs is to identify compounds that act through allosteric mechanisms, modulating the receptor by binding to a site different from the highly conserved orthosteric binding site.
• Allosteric modulators of mGluR2 were shown to be active in fear-potentiated startle, and in stress-induced hyperthermia models of anxiety. Furthermore, such compounds were shown to be active in reversal of ketamine- or amphetamine-induced
• BINA metabotropic glutamate receptor subtype 2 biphenyl-indanone
• Positive allosteric modulators enable potentiation of the glutamate response, but they have also been shown to potentiate the response to orthosteric mGluR2 agonists such as LY379268 or DCG-IV. These data provide evidence for yet another novel therapeutic approach to treat the above mentioned neurological and psychiatric diseases involving mGluR2, which would use a combination of a positive allosteric modulator of mGluR2 together with an orthosteric agonist of mGluR2.
• WO2010/130424, WO2010/130423 and WO2010/130422 published on 18 November 2010, disclose mGluR2 positive allosteric modulators.
• PET positron emission tomography
• PET Positron Emission Tomography
• radionuclides such as, for example, 15 O, 13 N, 11 C and 18 F for detection.
• positron emission tomography radiotracers have been reported so far for in vivo imaging of mGluRl and mGluR5. Up to our knowledge there is not any PET ligand that has been disclosed for imaging mGluR2 so far.
• the present invention relates to a compound having the Formula (I)
• R 1 is selected from the group consisting of cyclopropylmethyl and Ci
• R 2 is selected from chloro and trifluoromethyl
• R 3 is fluoro
• n is selected from 0, 1 and 2;
• the invention also relates to precursor compounds for the synthesis of a compound of formula (I) as previously defined.
• the present invention also relates to a compound of formula (V)
• R 1 is selected from the group consisting of cyclopropylmethyl and Ci- 3 alkyl substituted with one or more fluoro substituents;
• R 2 is selected from chloro and trifluoromethyl
• R 3 is fluoro
• n is selected from 0, 1 and 2;
• the invention also relates to reference materials, corresponding to the [ 12 C]- compounds of formula (I).
• the invention relates to novel compounds selected from the group consisting of
• Illustrative of the invention is a sterile solution comprising a compound of Formula (I) described herein.
• Exemplifying the invention is a use of a compound of formula (I) as described herein, for, or a method of, imaging a tissue, cells or a host, in vitro or in vivo.
• Further exemplifying the invention is a method of imaging a tissue, cells or a host, comprising contacting with or administering to a tissue, cells or a host, a compound of Formula (I) as described herein, and imaging the tissue, cells or host with a positron- emission tomography imaging system.
• the invention refers to a process for the preparation of a compound according to Formula (I) as described herein, wherein the C in the methoxy group is radiolabelled, herein referred to as [ U C]-(I), comprising the step of reacting a compound according to formula (V) as described herein, with [ U C]CH 3 I or
• the present invention is directed to compounds of formula (I) as defined herein before, and pharmaceutically acceptable salts thereof.
• the present invention is also directed to precursor compounds of formula (V), used in the synthesis of compounds of formula (I).
• R 1 is selected from cyclopropylmethyl and 2,2,2-trifluoroethyl; and R 2 is selected from chloro and trifluoromethyl.
• R 1 is cyclopropylmethyl and R 2 is chloro.
• n is 0 or 2.
• the invention relates to a compound according to formula [ U C]-(I)
• R 1 is selected from the group consisting of cyclopropylmethyl and Ci- 3 alkyl substituted with one or more fluoro substituents;
• R 2 is selected from chloro and trifluorom ethyl
• R 3 is fluoro
• n is selected from 0, 1 and 2;
• R 1 is selected from cyclopropylmethyl and 2,2,2- trifluoroethyl; and R 2 is selected from chloro and trifluorom ethyl.
• R 1 is cyclopropylmethyl and R 2 is chloro.
• n is 0 or 2.
• An additional embodiment of the invention relates to compounds wherein n is 2.
• R 1 and R 2 are as previously defined.
• the compound of Formula (I) as previously described is selected from the group consisting of
• the compound of Formula (V) as previously described is selected from the group consisting of 2- [ 1 - [8-chloro-3 -(cyclopropylmethyl)- 1 ,2,4-triazolo [4, 3 -a]pyridin-7-yl] -4- piperidinyl] -3 -fluoro-phenol,
• the compounds of Formula (I) and compositions comprising the compounds of Formula (I) can be used for imaging a tissue, cells or a host, in vitro or in vivo.
• the invention relates to a method of imaging or quantifying the mGluR2 receptor in a tissue, cells or a host in vitro or in vivo.
• the cells and tissues are preferably central nervous system cells and tissues in which the mGluR2 receptors are abundant.
• the mGluR2 receptor is abundant in central nervous system tissue, more in particular, in central nervous system tissue forming the brain; more in particular, forming the cerebral cortex, thalamic regions, accessory olfactory bulb, hippocampus, amygdala, caudate-putamen and nucleus accumbens.
• the host is a mammal.
• the compound of Formula (I) is administered intravenously, for example, by injection with a syringe or by means of a peripheral intravenous line, such as a short catheter.
• the compound of Formula (I) or a sterile solution comprising a compound of Formula (I) may in particular be administered by intravenous administration in the arm, into any identifiable vein, in particular in the back of the hand, or in the median cubital vein at the elbow.
• the invention relates to a method of imaging a tissue or cells in a mammal, comprising the intravenous administration of a compound of Formula (I), as defined herein, or a composition comprising a compound of Formula (I) to the mammal, and imaging the tissue or cells with a positron-emission tomography imaging system.
• the invention relates to a method of imaging a tissue or cells in a human, comprising the intravenous administration of a compound of Formula (I), as defined herein, or a sterile formulation comprising a compound of Formula (I) to the human, and imaging the tissue or cells with a positron- emission tomography imaging system.
• the invention relates to a method of imaging or quantifying the mGluR2 receptor in a mammal, comprising the intravenous
• the invention relates to the use of a compound of Formula (I) for imaging a tissue, cells or a host, in vitro or in vivo, or the invention relates to a compound of Formula (I), for use in imaging a tissue, cells or a host in vitro or in vivo, using positron-emission tomography.
• Ci- 3 alkyl shall denote a straight or branched saturated alkyl group having 1, 2 or
• Ci- 3 alkyl substituted with one or more fluoro substituents shall denote Ci-3alkyl as previously defined, substituted with 1, 2 or 3 or where possible, with more fluoro atoms.
• composition is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.
• the invention includes all stereoisomers of the compound of Formula (I) either as a pure stereoisomer or as a mixture of two or more stereoisomers.
• Enantiomers are stereoisomers that are non-superimposable mirror images of each other.
• a 1 : 1 mixture of a pair of enantiomers is a racemate or racemic mixture.
• Diastereomers or diastereoisomers
• Diastereomers are stereoisomers that are not enantiomers, i.e. they are not related as mirror images. Therefore, the invention includes enantiomers, diastereomers, racemates, and mixtures thereof.
• the absolute configuration may be specified according to the Cahn-Ingold-Prelog system.
• the configuration at an asymmetric atom may be specified by either R or S.
• Addition salts of the compounds according to Formula (I) and of the compounds of Formula (V) can also form stereoisomeric forms and are also intended to be
• Acceptable salts of the compounds of formula (I) are those wherein the counterion is pharmaceutically acceptable. However, salts of acids and bases which are non- pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. All salts, whether
• the pharmaceutically acceptable salts are defined to comprise the therapeutically active non-toxic acid addition salt forms that the compounds according to Formula (I) are able to form.
• Said salts can be obtained by treating the base form of the compounds according to Formula (I) with appropriate acids, for example inorganic acids, for example hydrohalic acid, in particular hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid and phosphoric acid; organic acids, for example acetic acid, hydroxyacetic acid, propanoic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzensulfonic acid, p-toluenesulfonic acid, cyclamic acid, salicylic acid, p-amino salicylic
• salt forms can be converted into the free base form by treatment with an appropriate base.
• some of the compounds of the present invention may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are also intended to be encompassed within the scope of this invention.
• the term "host” refers to a mammal, in particular to humans, mice, dogs and rats.
• the term “cell” refers to a cell expressing or incorporating the mGlu2 receptor.
• the compounds according to the invention can generally be prepared by a succession of steps, each of which is known to the skilled person.
• the compounds can be prepared according to the following synthesis methods.
• a final compound according to Formula [ C]-(I) wherein all variables are as previously defined can be prepared following art known procedures by cyclization of an intermediate compound of Formula (II) in the presence of a halogenating agent such as for example POCl 3 in a suitable solvent such as, for example, CH 3 CN or DCE, stirring the r.m. at a suitable temperature, using conventional heating or under microwave irradiation for the required time to achieve completion of the reaction, typically at 150 - 160°C for 5-15 min in a microwave oven.
• a halogenating agent such as for example POCl 3
• a suitable solvent such as, for example, CH 3 CN or DCE
• an intermediate compound of formula (III) can be reacted with an intermediate compound of formula (IV) in a suitable reaction-inert solvent such as, for example, toluene, in the presence of a suitable base such as, for example, CS2CO3, a metal-based catalyst, specifically a palladium catalyst, such as palladium(II) acetate, and a suitable ligand, such as for example BINAP, heating for a suitable period of time that allows the completion of the reaction, typically at 100-125 °C overnight in a sealed tube.
• a suitable reaction-inert solvent such as, for example, toluene
• a suitable base such as, for example, CS2CO3
• a metal-based catalyst specifically a palladium catalyst, such as palladium(II) acetate
• a suitable ligand such as for example BINAP
• an intermediate compound (III) can be reacted with an intermediate compound (IV) in the presence of a base, such as for example DIPEA, NaHC03 or CS2CO3, in a suitable inert solvent such as, for example, CH 3 CN or propionitrile, stirring the r.m. at a suitable temperature, using conventional heating or under microwave irradiation for the required time to achieve completion of the reaction, typically at 190-230°C for 15-30 min in a microwave oven, to yield a compound of Formula (I).
• a base such as for example DIPEA, NaHC03 or CS2CO3
• a suitable inert solvent such as, for example, CH 3 CN or propionitrile
• radiolabelling with radioactive carbon- 11 of compounds of formula [ 12 C]-(I) may be performed using radiochemical techniques well known to those skilled in the art, as shown in scheme 3.
• a [ u C]-methoxy group can be incorporated by reaction of a suitable phenolic precursor of formula (V) with [ U C]CH 3 I or [ u C]CH 3 OTf in the presence of a base, such as for example Cs 2 C0 3 , in an inert solvent such as for example DMF, stirring the r.m. at a suitable temperature using conventional heating or under microwave irradiation, for a suitable period of time to allow completion of the reaction, typically with conventional heating at 90°C for 3 min, followed by semi-preparative FIPLC purification.
• a base such as for example Cs 2 C0 3
• an inert solvent such as for example DMF
• Intermediate compounds according to Formula (II) can be prepared by art known procedures by reacting an intermediate of Formula (VI) with an acid halide of formula (Vila), which is commercially available, as shown in scheme 4.
• the reaction can be carried out using an inert-solvent such as for example DCM in the presence of a base such as for example Et 3 N, typically at r.t. for a suitable period of time to allow completion of the reaction.
• an inert-solvent such as for example DCM
• a base such as for example Et 3 N
• intermediate compounds according to Formula (II) can be prepared, following standard conditions that are known to those skilled in the art, by reacting an intermediate of Formula (VI) with a commercially available carboxylic acid of Formula (Vllb) via an amide bond formation reaction in the presence of a suitable coupling reagent.
• Intermediate compounds according to Formula (VI) can be prepared by reacting an intermediate compound of Formula (VIII) with hydrazine-hydrate according to reaction scheme 5.
• an intermediate compound (VIII) and hydrazine-hydrate are mixed in a suitable reaction-inert solvent, such as, for example, EtOH or THF and the mixture is stirred at a suitable temperature using conventional heating or under microwave irradiation, for a suitable period of time to allow completion of the reaction, typically at 160 °C under microwave irradiation for 20-40 min.
• a suitable reaction-inert solvent such as, for example, EtOH or THF
• an intermediate of Formula (III) can be reacted with an intermediate compound of Formula (IX) in a suitable reaction-inert solvent, such as, for example, CH 3 CN, in the presence of a suitable base, such as, for example, DIPEA, heating the r.m. at a suitable temperature, using conventional heating or under microwave irradiation for the required time to achieve completion of the reaction, typically at 190°C for 20 min in a microwave oven.
• halo is chloro, bromo or iodo.
• Intermediate compounds of formula (IX) can be prepared by described synthesis methods well known to the person skilled in the art, such as, for example, by the reaction sequence shown in scheme 7 for intermediates wherein R 2 is chlorine, hereby named (IX-a).
• 2,3-dichloropyridine can be treated with an alkyl-lithium derivative, such as for example «-BuLi, in a suitable inert and dry solvent, such as for example Et 2 0 or THF, and reacted with the desired halogenating agent (halo 2 ), such as for example iodine, stirring the r.m. at a suitable temperature for the required time to achieve completion of the reaction, typically at -78°C to r.t. overnight.
• halo 2 halogenating agent
• reaction of an intermediate of Formula (X) with a suitable trifluoromethylating agent such as for example fluorosulfonyl(difluoro)acetic acid methyl ester
• a suitable reaction-inert solvent such as, for example, DMF
• a suitable coupling agent such as for example, copper iodide
• thermal conditions such as, for example, heating the r.m. at 160 °C under microwave irradiation for 45 min, to afford intermediate of formula (IX-b).
• a commercially available 2-chloro-4-halopyridine can be reacted with a strong base such as, for example, n-BuLi, and further treated with an iodinating agent such as, for example, iodine.
• This reaction is performed in a suitable reaction-inert solvent such as, for example, THF at low temperature for a period of time that allows the completion of the reaction, typically at -78 °C for 2 h.
• Intermediate compounds of formula (III) can be prepared by a two step synthesis well known to the person skilled in the art, such as, for example, by the reaction sequence shown in scheme 10.
• a compound of formula (XI) can be subjected first to a hydrogeno lysis reaction, in a suitable inert solvent in the presence of a catalyst such as, for example, 5% or 10% palladium on activated carbon, for a period of time that ensures the completion of the reaction, typically at 100 °C and 1 atmosphere of hydrogen in an H- cube apparatus.
• this intermediate can be deprotected with HC1 in iPrOH or TFA in DCM, at a suitable temperature, typically r.t., for a period of time to allow cleavage of the BOC protecting group, typically 2 h.
• These two steps can be also reversed: first deprotection and then hydrogenation to give intermediate compound of formula (III).
• an intermediate compound of formula (XII) can be reacted with N-Boc- 1,2,3, 6- tetrahydropyridine-4-boronic acid pinacol ester, available from commercial sources, in the presence of a palladium(O) catalyst, such as, for example, Pd(PPh 3 ) 4 , and in the presence of a base, such as, for example, K 2 C0 3 or Cs 2 C0 3 , in a suitable inert solvent such as, for example, dioxane, stirring the r.m. at a suitable temperature using conventional heating or under microwave irradiation for the required time to achieve completion of the reaction, typically at 150°C for 10 min in a microwave oven.
• Intermediate compounds according to formula (XII) are either commercially available or can be prepared by synthesis methods well known by the skilled person, such as, for example, by the reaction sequence shown in scheme 12.
• an intermediate compound of formula (XIII) can be reacted with a methylating reagent, such as, for example, CH 3 I, in the presence of a suitable base, such as, for example, K 2 C0 3 or Cs 2 C0 3 , in a reaction-inert solvent, such as for example, CH 3 CN, stirring the r.m. at a suitable temperature using conventional heating or under microwave irradiation for the required period of time to achieve completion of the reaction, typically at 150°C for 10 min in a microwave oven.
• a methylating reagent such as, for example, CH 3 I
• a suitable base such as, for example, K 2 C0 3 or Cs 2 C0 3
• a reaction-inert solvent such as for example, CH 3 CN
• a phenolic intermediate of formula (XIV) can be brominated in ortho position to the hydroxyl with a brominating reagent, such as, for example, bromine or BS, in the presence of an aliphatic amine, such as, for example, tert-butylamine, in a suitable inert solvent, such as, for example, DCM, stirring the r.m. at low temperature, typically at - 10°C or -40°C, for the required period of time to achieve completion of the reaction, typically 30 min.
• a brominating reagent such as, for example, bromine or BS
• an aliphatic amine such as, for example, tert-butylamine
• a suitable inert solvent such as, for example, DCM
• an intermediate compound of formula (IV) can be prepared following art known procedures by cyclization of an intermediate compound of Formula (XV) in the presence of an halogenating agent such as for example POCl 3 in a suitable solvent such as, for example, DCE, stirred under microwave irradiation, for a suitable period of time that allows the completion of the reaction, as for example 5 min at a temperature between 140-200 °C.
• an halogenating agent such as for example POCl 3
• a suitable solvent such as, for example, DCE
• intermediate compounds of formula (IV) can be prepared following art known procedures, as shown in scheme 15, by cyclization of an intermediate compound of formula (XVI) after heating for a suitable period of time to allow the completion of the reaction, as for example 1 h at a temperature between 140-200 °C.
• reaction schemes 14 and 15 all variables are defined as in Formula (I) and halo is chloro, bromo or iodo.
• an intermediate compound of formula (XVII) can react with acid halides of formula (Vila) in an inert- solvent, such as for example DCM, in the presence of a base such as for example Et 3 N, usually at r.t. for a suitable period of time that allows completion of the reaction, for example 20 min, to yield an intermediate compound of formula (XV).
• an inert- solvent such as for example DCM
• a base such as for example Et 3 N
• an intermediate of formula (XVI) can be prepared by reaction of intermediate compounds of formula (XVIII) with acid halides of formula (Vila).
• the reaction can be carried out using an inert-solvent such as for example DCM in the presence of a base such as for example Et 3 N, typically at r.t, for a suitable period of time that allows completion of the reaction, typically for 20 min.
• an intermediate compound of formula (IX) can be reacted with hydrazine in a suitable reaction-inert solvent, such as, for example, EtOH, THF or 1,4-dioxane at a suitable temperature using conventional heating or under microwave irradiation for the required period of time to achieve completion of the reaction, typically at 160°C under microwave irradiation for 30 min, or by classical thermal heating at 70°C overnight.
• a suitable reaction-inert solvent such as, for example, EtOH, THF or 1,4-dioxane
• an intermediate compound of formula (XVII) can be prepared by reacting an intermediate compound of formula (XIX) with hydrazine in a suitable reaction-inert solvent, such as, for example, EtOH, THF or 1,4-dioxane at a suitable temperature using conventional heating or under microwave irradiation for the required period of time to achieve completion of the reaction, typically at 160°C under microwave irradiation for 30 min, or by classical thermal heating at 70°C overnight.
• a suitable reaction-inert solvent such as, for example, EtOH, THF or 1,4-dioxane
• an intermediate compound of formula (IX) can be reacted with benzyl alcohol in a suitable reaction-inert solvent, such as, for example, DMF in the presence of a suitable base, such as for example NaH at r.t., for a suitable period of time that allows the completion of the reaction, typically for 1 h.
• a suitable reaction-inert solvent such as, for example, DMF
• a suitable base such as for example NaH at r.t.
• intermediate compounds of formula (V) can also be synthesized by a reaction sequence as shown in scheme 22.
• an intermediate compound of formula (XX) can be reacted with an intermediate compound of formula (IV) in the presence of a suitable base, such as, for example, NaHC0 3 , in an inert solvent such as, for example, CH 3 CN, propionitrile or butyronitrile, stirring the r.m. at a suitable temperature, using conventional heating or under microwave irradiation for the required period of time to achieve completion of the reaction, typically at 180 - 230°C for 10-30 min in a microwave oven, or for 1.5 - 16 h using conventional heating in a sealed tube.
• a suitable base such as, for example, NaHC0 3
• an inert solvent such as, for example, CH 3 CN, propionitrile or butyronitrile
• a compound of formula (XXI) can be subjected to a hydrogenolysis reaction, in a suitable inert solvent in the presence of a catalyst such as, for example, 5% or 10% palladium on activated carbon, for a period of time that ensures the completion of the reaction, typically at 100 °C and 1 atmosphere of hydrogen in an H-cube® apparatus.
• a catalyst such as, for example, 5% or 10% palladium on activated carbon
• an intermediate compound according to formula (XXII) can be reacted with a diluted solution of an acid, such as, for example, HCl in iPrOH or TFA in DCM, at a suitable temperature, typically r.t, for a period of time to allow cleavage of the Boc protecting group, typically 2 h.
• an acid such as, for example, HCl in iPrOH or TFA in DCM
• an intermediate compound of formula (XXIII) can be reacted with N-Boc- l,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester, available from commercial sources, in the presence of a palladium(O) catalyst, such as, for example, Pd(PPh 3 ) 4 , and in the presence of a base, such as, for example, K 2 C0 3 or Cs 2 C0 3 , in a suitable inert solvent such as, for example, dioxane, stirring the r.m. at a suitable temperature using conventional heating or under microwave irradiation for the required time to achieve completion of the reaction, typically at 150°C for 10 min in a microwave oven.
• a palladium(O) catalyst such as, for example, Pd(PPh 3 ) 4
• a base such as, for example, K 2 C0 3 or Cs 2 C0 3
• a suitable inert solvent such as, for example, dioxane
• an intermediate compound of formula (XIII) can be reacted with benzyl bromide, in the presence of a suitable base such as, for example, K 2 C0 3 or Cs 2 C0 3 , in an inert solvent such as, for example, CH 3 CN, stirring the r.m. at a suitable temperature using conventional heating or under microwave irradiation for the required time to achieve completion of the reaction, typically at 150°C for 10 min in a microwave oven.
• a suitable base such as, for example, K 2 C0 3 or Cs 2 C0 3
• an inert solvent such as, for example, CH 3 CN
• the compounds according to the present invention find various applications for imaging tissues, cells or a host, both in vitro and in vivo. Thus, for instance, they can be used to map the differential distribution of mGluR2 in subjects of different age and sex. Further, they allow one to explore for differential distribution of mGluR2 in subjects afflicted by different diseases or disorders. Thus, abnormal distribution may be helpful in diagnosis, case finding, stratification of subject populations, and in monitoring disease progression in individual subjects.
• the radioligands may further find utility in determining mGluR2 site occupancy by other ligands. Since the radioligand is administered in trace amounts, no therapeutic effect may be attributed to the administration of the radioligands according to the invention. Experimental Part
• LCMS liquid chromatography/mass spectrometry
• GCMS gas chromatography/mass spectrometry
• HPLC high- performance liquid chromatography
• aq means aqueous
• Boc'V'BOC means tert- butoxycarbonyl
• nBuLi means «-butyllithium
• DCE means 1,2-dichloroethane
• DCM means dichloromethane
• DMF means N,N-dimethylformamide
• EtOH means ethanol
• EtOAc means ethyl acetate
• THF means tetrahydrofuran
• DIPE means diisopropyl ether
• DIPEA means diisopropylethyl amine
• Et 3 N means triethylamine
• BINAP means l,l'-[l,r-binaphthalene]-2,2'
• Microwave assisted reactions were performed in a single-mode reactor: Biotage InitiatorTM Sixty microwave reactor (Biotage) or in a multimode reactor: MicroSYNTH Labstation (Milestone, Inc.). Hydrogenation reactions were performed in a continuous flow hydrogenator H-CUBE from ThalesNano Nanotechnology Inc.
• TLC Thin layer chromatography
• silica gel 60 F254 plates Merck
• Open column chromatography was performed on silica gel, mesh 230-400 particle size and 60 A pore size (Merck) under standard techniques.
• Automated flash column chromatography was performed using ready-to-connect cartridges from Merck, on irregular silica gel, particle size 15-40 ⁇ (normal phase disposable flash columns) on an SPOT or LAFLASH system from Armen Instrument.
• intermediate 1-14 (0.20 g, 0.60 mmol) in MeOH (1 mL). The mixture was stirred at r.t. for 1.5 h. The mixture was diluted with Na 2 CC>3 (aq. sat. sol.) and extracted with DCM. The organic phase was separated, dried (Na 2 S0 4 ) and the solvent evaporated in vacuo to yield intermediate 1-15 (0.12 g, 85%).
• Intermediate 1-16 was synthesized following the same methodology described for 1-13: starting from 2-Bromo-3-fluoroanisole [C.A.S. 446-59-3] (3.18g, 15.82 mmol) and 3,6- dihydro-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l(2H)-pyridinecarboxylic acid, 1, 1-dimethylethyl ester [C.A.S. 286961-14-6], (4 g, 12.9 mmol) to yield intermediate 1-16 (6.63 g, quant, yield).
• Intermediate 1-18 was synthesised as reported for intermediate 1-12. Starting from 2- Bromo-3,5-difluorophenol (0.5 g, 2.39 mmol) and Mel (0.22 mL, 3.58 mmol) to yield intermediate 1-18 (0.53 g, quant, yield).
• Intermediate 1-19 was synthesized following the same methodology described for 1-13: starting from intermediate 1-18 (0.53 g, 2.39 mmol) and 3,6-dihydro-4-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)-l(2H)-pyridinecarboxylic acid 1, 1-dimethylethyl ester [C.A.S. 286961-14-6] (0.62 g, 1.99 mmol) to yield intermediate 1-19 (1.2 g quant, yield).
• Intermediate 1-25 was synthesized as reported for intermediate 1-24: starting from intermediate 1-17 (3.4 g, 7.41 mmol) and treated with HC1 (7M in iPrOH) (23.5 mL), intermediate 1-25 was obtained (1.7 g, quant, yield).
• Intermediate 1-26 was synthesized as reported for intermediate 1-24: starting from intermediate 1-19 (1.2 g, 1.99 mmol) and treated with HC1 (7M in iPrOH) (4 mL), intermediate 1-26 was obtained (0.33 g, 73.5%).
• Intermediate 1-28 was synthesized as reported for intermediate 1-14: starting from intermediate 1-23 (0.54 g, 1.66 mmol) that was reduced to yield intermediate 1-28 (0.54 g, quant, yield).
• Intermediate 1-32 was synthesized following the same methodology described for 1-29: starting from intermediate 1-27 that was reduced by hydrogenation to yield intermediate 1-32 (0.293 g, 84.4%).
• Intermediate 1-41 was synthesised following the same methodology described for 1-34: starting from intermediate 1-40 (0.94 g, 4.49 mmol) and benzyl bromide [C.A.S. 100- 39-0] (0.53 mL, 4.49 mmol) to yield intermediate 1-41 (1.18 g, 88%).
• Intermediate 1-43 was synthesized as described for intermediate 1-35. Starting from intermediate 1-38 (1.48 g, 5.26 mmol) coupled with 3,6-dihydro-4-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)-l(2H)-pyridinecarboxylic acid, 1, 1-dimethylethyl ester [C.A.S. 286961-14-6] (1.36 g, 4.39 mmol) to yield intermediate 1-43 (1.5 g, 85%).
• Intermediate 1-44 was synthesized as described for intermediate 1-35. Starting from intermediate 1-39 (1.06 g, 3.77 mmol) coupled with 3,6-dihydro-4-(4,4,5,5-tetramethyl- l,3,2-dioxaborolan-2-yl)-l(2H)-pyridinecarboxylic acid, 1, 1-dimethylethyl ester
• Intermediate 1-45 was synthesized as described for intermediate 1-35. Starting from intermediate 1-41 (1.18 g, 3.96 mmol) coupled with 3,6-dihydro-4-(4,4,5,5-tetramethyl- l,3,2-dioxaborolan-2-yl)-l(2H)-pyridinecarboxylic acid, 1, 1-dimethylethyl ester
• Intermediate 1-46 was synthesized as described for intermediate 1-35. Starting from intermediate 1-42 (1.43 g, 4.78 mmol) coupled with 3,6-dihydro-4-(4,4,5,5-tetramethyl- l,3,2-dioxaborolan-2-yl)-l(2H)-pyridinecarboxylic acid, 1, 1-dimethylethyl ester
• Intermediate 1-48 was synthesized as described for intermediate 1-36. Starting from 1-44 (1 g, 2.63 mmol) and treated with HC1 (7 M in iPrOH) (5 mL), intermediate 1-48 was obtained (0.46 g, 62%).
• Intermediate 1-49 was synthesized as described for intermediate 1-36. Starting from 1-45 (0.9 g, 2.24 mmol) and treated with HC1 (7 M in iPrOH) (5 mL), intermediate 1-49 was obtained (0.38 g, 56.6%). Intermediate 1-50
• Intermediate 1-50 was synthesized as described for intermediate 1-36. Starting from intermediate 1-46 (1.51 g, 3.76 mmol) and treated with HC1 (7 M in iPrOH) (7.5 mL), intermediate 1-50 was obtained (1.07 g, 94%). Intermediate 1-51
• Intermediate 1-51 was synthesized following the same methodology described for 1-37: Starting from intermediate 1-47 (1.1 g, 3.88 mmol) through a hydrogenation, intermediate 1-51 (0.75 g, 98%) was obtained.
• Intermediate 1-53 was synthesized following the same methodology described for 1-37: Starting from intermediate 1-49 (0.38 g, 1.27 mmol) through a hydrogenation, intermediate 1-53 (0.271 g, quant, yield) was obtained.
• Intermediate 1-59 was synthesized following the same synthetic procedure described for intermediate 1-58. Starting from intermediate 1-4 (0.1 g, 0.3 mmol) and 1-52 (0.087 g, 0.45 mmol), derivative 1-59 was obtained (0.034 g, 28.3%). C 21 H 22 CIFN 4 O. LCMS: Rt 2.76, m/z 401 [(M + H)] + (method 3).1H NMR (500 MHz, DMSO-i ) ⁇ ppm 0.21 - 0.34 (m, 2 H), 0.45 - 0.56 (m, 2 H), 1.05 - 1.21 (m, 1 H), 1.67 (br.
• Intermediate 1-60 was synthesized following the same synthetic procedure described for intermediate 1-58. Starting from intermediates 1-4 (0.1 g, 0.3 mmol) and 1-53 (0.1 g, 0.45 mmol), intermediate 1-60 was obtained (0.016 g, 11.6%). C 21 H 21 CIF 2 N 4 O. LCMS: Rt 2.85, m/z 419 [(M + H)] + (method 3). 1 H MR (500 MHz, DMSO-i ) ⁇ ppm 0.21 - 0.33 (m, 2 H), 0.44 - 0.56 (m, 2 H), 1.12 - 1.21 (m, 1 H), 1.71 (br.
• Intermediate 1-61 was synthesized following the same synthetic procedure described for intermediate 1-58. Starting from intermediate 1-4 (0.1 g, 0.3 mmol) and 1-54 (0.17 g, 0.6 mmol), intermediate 1-61 was obtained (0.014 g, 11.2%). C 21 H 21 CIF 2 N 4 O.
• Intermediate 1-67 was synthesized following the same approach described for intermediate 1-64. Starting from 1-29 (0.35 g, 1.67 mmol) and 1-1 (0.46 g, 1.67 mmol), intermediate 1-67 was obtained (0.21 g, 35.5%).
• Intermediate 1-68 was synthesized following the same approach described for intermediate 1-65. Starting from 1-67 (0.21 g, 0.59 mmol) and hydrazine hydrate (0.57, 11.88 mmol), intermediate 1-68 was obtained (0.11 g, 52.3%). Intermediate 1-69
• Intermediate 1-69 was synthesized following the same approach reported for intermediate 1-66. Starting from intermediate 1-68 (0.11 g, 0.31 mmol) and 3,3,3- trifluoropropionyl chloride [C.A.S. 41463-83-6] (0.065 mL, 0.47 mmol), intermediate 1-69 (0.144 g, quant, yield) was obtained. B. Preparation of the final compounds
• Compound B-7 was synthesized following the same approach described for B-2.
• HPLC analysis was performed on a LaChrom Elite HPLC pump (Hitachi, Darmstadt, Germany) connected to a UV spectrometer (Hitachi) set at 254 nm.
• the HPLC eluate after passage through the UV detector was led over a 7.62 cm (3 inch) Nal(Tl) scintillation detector connected to a single channel analyzer (Medi-Laboratory Select, Mechelen, Belgium).
• radioactivity measurements during biodistribution studies and in vivo stability analyses were done using an automatic gamma counter (with a 3 in. Nal(Tl) well crystal) coupled to a multichannel analyzer (Wallac 1480 Wizard 3", Wallac, Turku, Finland).
• Carbon- 11 was produced using a Cyclone 18/9 cyclotron (Ion Beam
• the target gas which was a mixture of N 2 (95 %) and H 2 (5 %) was irradiated using 18
• [ U C]CH 4 was then reacted with vaporous I 2 at 650 °C to convert it to [ n C]methyl iodide ([ u C]MeI).
• the resulting volatile [ u C]MeI was bubbled with a flow of helium through a solution of radiolabeling precursor 1-58 (for [ u C]B-4 ), 1-59 (for [ n C]B-6, 1- 62 (for [ u C]B-2), 1-61 (for [ u C]B-7), 1-60 (for [ U C]B-10), 1-63 (for [ u C]B-3) (0.2 mg) and Cs 2 C0 3 (1-3 mg) in anhydrous DMF (0.2 mL).
• the reaction mixture was heated at 90 °C for 3 min. After dilution, the crude reaction mixture was injected onto an HPLC system consisting of a semi-preparative XBridge® column (Ci 8 , 5 ⁇ ; 4.6 mm x 150 mm; Waters, Milford, MA, USA) that was eluted with a mixture of 0.05 M sodium acetate buffer (pH 5.5) and EtOH (50:50 v/v) at a flow rate of 1 mL/min. UV detection was done at 254 nm. The radiolabeled product was collected between 12 and 16 min (small difference in R t time for the different tracers).
• the collected peak corresponding to the desired radioligand was then diluted with saline (Mini Plasco ® , Braun, Melsungen, Germany) to obtain a final EtOH concentration of 10 % and the solution was sterile filtered through 0.22 ⁇ membrane filter (Millex ® -GV, Millipore, Ireland). This formulation was then used for all in vivo experiments.
• radiotracers The identity of the radiotracers was confirmed using the same analytical HPLC method as described above after co-injection with their non-radioactive analogue.
• Values are peak values, and are obtained with experimental uncertainties that are commonly associated with this analytical method.
• melting points were determined in open capillary tubes on a Mettler FP62 apparatus. Melting points were measured with a temperature gradient of 10 °C/minute. Maximum temperature was 300 °C. The melting point was read from a digital display.
• the HPLC measurement was performed using an HP 1100 (Agilent Technologies) system comprising a pump (quaternary or binary) with degasser, an autosampler, a column oven, a diode-array detector (DAD) and a column as specified in the respective methods below.
• Flow from the column was split to the MS spectrometer.
• the MS detector was configured with either an electrospray ionization source or an ESCI dual ionization source (electrospray combined with atmospheric pressure chemical ionization). Nitrogen was used as the nebulizer gas.
• the source temperature was maintained at 140 °C. Data acquisition was performed with MassLynx-Openlynx software.
• the UPLC (Ultra Performance Liquid Chromatography) measurement was performed using an Acquity UPLC (Waters) system comprising a sampler organizer, a binary pump with degasser, a four column's oven, a diode-array detector (DAD) and a column as specified in the respective methods below. Column flow was used without split to the MS detector.
• the MS detector was configured with an ESCI dual ionization source (electrospray combined with atmospheric pressure chemical ionization). Nitrogen was used as the nebulizer gas. The source temperature was maintained at 140 °C. Data acquisition was performed with MassLynx-Openlynx software.
• Reversed phase UPLC was carried out on a BEH-C18 column (1.7 ⁇ , 2.1 x 50 mm) from Waters, with a flow rate of 0.8 ml/min, at 60°C without split to the MS detector.
• the gradient conditions used are: 95 % A (0.5 g/1 ammonium acetate solution + 5 % acetonitrile), 5 % B (mixture of acetonitrile / methanol, 1/1), to 20 % A, 80 % B in 4.9 minutes, to 100 % B in 5.3 minutes, kept till 5.8 minutes and equilibrated to initial conditions at 6.0 minutes until 7.0 minutes.
• Injection volume 0.5 ⁇ .
• Low-resolution mass spectra (single quadrupole, SQD detector) were acquired by scanning from 100 to 1000 in 0.1 seconds using an inter- channel delay of 0.08 second.
• the capillary needle voltage was 3 kV.
• the cone voltage was 20 V for positive ionization mode and 30 V for negative ionization mode.
• BEH-C18 column (1.7 ⁇ , 2.1 x 50 mm) from Waters, with a flow rate of 0.8 ml/min, at 60°C without split to the MS detector.
• the gradient conditions used are: 95 % A (0.5 g/1 ammonium acetate solution + 5 % acetonitrile), 5 % B (mixture of acetonitrile / methanol, 1/1), kept 0.2 minutes, to 20 % A, 80 % B in 3.5 minutes, to 100 % B in 3.8 minutes, kept till 4.15 minutes and equilibrated to initial conditions at 4.3 minutes until 5.0 minutes.
• Injection volume 0.5 ⁇ .
• Low-resolution mass spectra (single quadrupole, SQD detector) were acquired by scanning from 100 to 1000 in 0.1 seconds using an inter-channel delay of 0.08 second.
• the capillary needle voltage was 3 kV.
• the cone voltage was 20 V for positive ionization mode and 30 V for negative ionization mode.
• BEH-C18 column (1.7 ⁇ , 2.1 x 50 mm) from Waters, with a flow rate of 1.0 ml/min, at 50°C without split to the MS detector.
• the gradient conditions used are: 95 % A (0.5 g/1 ammonium acetate solution + 5 % acetonitrile), 5 % B (acetonitrile), to 40 % A, 60 % B in 4.4 minutes, to 5 % A, 95 % B in 5.6 minutes, kept till 5.8 minutes and equilibrated to initial conditions at 6.0 minutes until 7.0 minutes.
• Injection volume 0.5 ⁇ .
• Low-resolution mass spectra (single quadrupole, SQD detector) were acquired by scanning from 100 to 1000 in 0.1 seconds using an inter-channel delay of 0.08 second.
• the capillary needle voltage was 3 kV.
• the cone voltage was 25 V for positive ionization mode and 30 V for negative ionization mode.
• Reversed phase UPLC was carried out on a BEH-C18 column (1.7 ⁇ , 2.1 x 50 mm) from Waters, with a flow rate of 1.0 ml/min, at 50°C without split to the MS detector.
• the gradient conditions used are: 95 % A (0.5 g/1 ammonium acetate solution + 5 % acetonitrile), 5 % B (acetonitrile), to 40 % A, 60 % B in 2.8 minutes, to 5 % A, 95 % B in 3.6 minutes, kept till 3.8 minutes and equilibrated to initial conditions at 4.0 minutes until 5.0 minutes.
• Injection volume 0.5 ⁇ .
• Low-resolution mass spectra (single quadrupole, SQD detector) were acquired by scanning from 100 to 1000 in 0.1 seconds using an inter-channel delay of 0.08 second.
• the capillary needle voltage was 3 kV.
• the cone voltage was 25 V for positive ionization mode and 30 V for negative ionization mode.
• Reversed phase HPLC was carried out on a Sunfire-C18 column (2.5 ⁇ , 2.1 x 30 mm) from Waters, with a flow rate of 1.0 ml/min, at 60°C.
• the gradient conditions used are: 95 % A (0.5 g/1 ammonium acetate solution + 5 % of acetonitrile), 2.5 % B (acetonitrile), 2.5 % C (methanol) to 50 % B, 50 % C in 6.5 minutes, kept till 7.0 minutes and equilibrated to initial conditions at 7.3 minutes until 9.0 minutes.
• High-resolution mass spectra (Time of Flight, TOF detector) were acquired by scanning from 100 to 750 in 0.5 seconds using a dwell time of 0.3 seconds.
• the capillary needle voltage was 2.5 kV for positive ionization mode and 2.9 kV for negative ionization mode.
• the cone voltage was 20 V for both positive and negative ionization modes.
• Leucine-Enkephaline was the standard substance used for the lock mass calibration.
• the compounds provided in the present invention are positive allosteric modulators of mGluR2. These compounds appear to potentiate glutamate responses by binding to an allosteric site other than the glutamate binding site.
• the response of mGluR2 to a concentration of glutamate is increased when compounds of Formula (I) are present.
• Compounds of Formula (I) are expected to have their effect substantially at mGluR2 by virtue of their ability to enhance the function of the receptor.
• the effects of positive allosteric modulators tested at mGluR2 using the [ 35 S]GTPyS binding assay method described below and which is suitable for the identification of such compounds, and more particularly the compounds according to Formula (I), is shown in Table II. r 35 SlGTPyS binding assay
• the [ 35 S]GTPyS binding assay is a functional membrane-based assay used to study G-protein coupled receptor (GPCR) function whereby incorporation of a non-hydrolysable form of GTP, [ 35 S]GTPyS (guanosine 5 '-triphosphate, labelled with gamma-emitting 35 S), is measured.
• GPCR G-protein coupled receptor
• the G-protein a subunit catalyzes the exchange of guanosine 5 '-diphosphate (GDP) by guanosine triphosphate (GTP) and on activation of the GPCR by an agonist, [ S]GTPyS, becomes incorporated and cannot be cleaved to continue the exchange cycle (Harper (1998) Current Protocols in Pharmacology 2.6.1-10, John Wiley & Sons, Inc.). The amount of radioactive [ 35 S]GTPyS
• mGluR2 receptors are shown to be preferentially coupled to God-protein, a preferential coupling for this method, and hence it is widely used to study receptor activation of mGluR2 receptors both in recombinant cell lines and in tissues.
• PAM positive allosteric modulation
• CHO-cells were cultured to pre-confluence and stimulated with 5 mM butyrate for 24 h. Cells were then collected by scraping in PBS and cell suspension was centrifuged (10 min at 4000 RPM in benchtop centrifuge). Supernatant was discarded and pellet gently resuspended in 50 mM Tris-HCl, pH 7.4 by mixing with a vortex and pipetting up and down. The suspension was centrifuged at 16,000 RPM (Sorvall RC-5C plus rotor SS-34) for 10 minutes and the supernatant discarded.
• the pellet was homogenized in 5 mM Tris-HCl, pH 7.4 using an ultra-turrax homogenizer and centrifuged again (18,000 RPM, 20 min, 4 °C). The final pellet was resuspended in 50 mM Tris-HCl, pH 7.4 and stored at -80 °C in appropriate aliquots before use. Protein concentration was determined by the Bradford method (Bio-Rad, USA) with bovine serum albumin as standard.
• Measurement of mGluR2 positive allosteric modulatory activity of test compounds was performed as follows. Test compounds and glutamate were diluted in assay buffer containing 10 mM HEPES acid, 10 mM HEPES salt, pH 7.4, 100 mM NaCl, 3 mM MgCl 2 and 10 ⁇ GDP. Human mGlu2 receptor-containing membranes were thawed on ice and diluted in assay buffer supplemented with 14 ⁇ g/ml saponin. Membranes were pre-incubated with compound alone or together with a predefined ( ⁇ EC 2 o) concentration of glutamate (PAM assay) for 30 min at 30°C. After addition of [ 35 S]GTPyS (f.c.
• concentration-response curves of representative compounds of the present invention -obtained in the presence of EC 20 of mGluR2 agonist glutamate to determine positive allosteric modulation (PAM)- were generated using the Lexis software interface (developed at J&J). Data were calculated as % of the control glutamate response, defined as the maximal response that is generated upon addition of glutamate alone. Sigmoid concentration-response curves plotting these percentages versus the log concentration of the test compound were analyzed using non-linear regression analysis. The concentration producing half-maximal effect is then calculated as EC 5 o- The pEC 5 o values below were calculated as the -log EC50, when the EC50 is expressed in M.
• pEC 5 o values were calculated from a concentration-response experiment of at least 8 concentrations. If more experiments were performed, the average pEC 5 o value is reported and error deviation was ⁇ 0.5.
• % ID percentage of injected dose
• % ID/g percentage of injected dose per gram tissue
• Table 1 shows the % ID values at 2 min, 30 min and 60 min p.i. of the radiotracer.
• the total initial brain uptake of the tracer was 0.88 % of the ID, with 0.69 % ID in the cerebrum and 0.17 % ID in the cerebellum.
• At 2 min p.i.4.3 % of the injected dose was present in the blood, and this cleared to 2.0 % by 60 min p.i.
• the tracer was cleared mainly by the hepatobiliary system as there was in total 35.7 % of ID present in liver and intestines 60 min after injection of the radiotracer.
• % ID/g tissue values were normalized for body weight.
• the normalized values (SUV, standard uptake value) for striatum, hippocampus, cortex and cerebellum are presented in Table 2.
• the radioactivity concentration has increased for all brain regions. This accumulation of radioactivity in all studied brain regions is consistent with the fact that mGluR2 receptors are expressed in several brain areas including hippocampus, cortical regions, olfactory bulb, cerebellum and striatum. Most significant increase was observed for striatum (SUV 1.22 at 2 min p.i. to SUV 2.14 at 30 min p.i.), followed by cerebellum. The highest radioactivity concentration at 30 min is found in the cerebellum (SUV 2.62), followed by striatum. For all brain regions the radioactivity concentration at 60 min p.i. is lower compared to 30 min time point, indicating that wash-out has started. Table 2. [ n C]B-2 concentration in different brain regions and blood at 2, 30 and 60 min p. i. normalized for the body weight of the animal
• Table 3 shows the % ID values at 2 min, 30 min and 60 min p.i. of the radiotracer. At 2 min p.i. 5.6 % of the ID was present in the blood, and this cleared to 3.3 % by 60 min after injection of the tracer. The total initial brain uptake of the tracer was 0.58 %, with 0.45 % of the ID in the cerebrum and 0.10 % in the cerebellum. At 60 min after injection of the radiotracer, 26.5 % ID was present in the liver and intestines.
• Hippocampus 0.010 ⁇ 0.001 0.026 ⁇ 0.007 0.021 ⁇ 0.005
• % ID/g tissue values were normalized for body weight.
• the normalized values for striatum, hippocampus, cortex and cerebellum are presented in Table 4.
• the radioactivity concentration has increased for all brain regions. This accumulation of radioactivity in all studied brain regions is consistent with the fact that mGluR2 receptors are expressed in several brain areas including hippocampus, cortical regions, olfactory bulb, cerebellum and striatum. Most significant increase was observed for striatum and cerebellum (SUV 1.46 at 2 min p.i. to SUV 2.31 at 30 min p.i.). The highest radioactivity concentration at 30 min is found in the cerebellum and the striatum SUV -2.32), followed by the cortex. For all brain regions the radioactivity concentration at 60 min p.i. is lower compared to 30 min time point, indicating that wash-out has started.
• Table 5 shows the % ID values at 2 min, 30 min and 60 min p.i. of the radiotracer. At 2 min p.i. 5.4 % of the ID was present in blood, and this cleared to 3.7 % by 60 min after injection of the tracer. The total initial brain uptake of the tracer was 0.75 %, with 0.53 % of the ID in the cerebrum and 0.18 % in the cerebellum. At 60 min after injection of the radiotracer, 28.7 % ID was present in the liver and intestines.
• Hippocampus 0.020 ⁇ 0.004 0.030 ⁇ 0.003 0.022 ⁇ 0.003
• % ID/g tissue values were normalized for body weight.
• the normalized values for striatum, hippocampus, cortex and cerebellum are presented in Table 6.
• the radioactivity concentration has increased for all brain regions. This accumulation of radioactivity in all studied brain regions is consistent with the fact that mGluR2 receptors are expressed in several brain areas including hippocampus, cortical regions, olfactory bulb, cerebellum and striatum. Most significant increase was observed for striatum and cortex (SUV -1.13 at 2 min p.i. to SUV -1.71 at 30 min p.i.). The highest radioactivity concentration at 30 min is found in the cerebellum (SUV 2.0), followed by the cortex. For all brain regions the radioactivity concentration at 60 min p.i. is lower compared to 30 min time point, indicating that wash-out has started.
• Table 7 shows the % ID values at 2 min, 30 min and 60 min p.i. of the radiotracer. At 2 min p.i. 6.5 % of the injected dose was present in the blood, and this cleared to 3.6 % by 60 min after injection of the tracer. The total initial brain uptake of the tracer was 0.65 %, with 0.45 % of the ID in the cerebrum and 0.17 % in the cerebellum. At 60 min after injection of the radiotracer, 30.6 % ID was present in the liver and intestines.
• % ID/g tissue values were normalized for body weight.
• the normalized values for striatum, hippocampus, cortex and cerebellum are presented in Table 8.
• the radioactivity concentration has increased for all brain regions. This accumulation of radioactivity in all studied brain regions is consistent with the fact that mGluR2 receptors are expressed in several brain areas including hippocampus, cortical regions, olfactory bulb, cerebellum and striatum. Most significant increase was observed for striatum (SUV -1.01 at 2 min p.i. to SUV -1.70 at 30 min p.i.). The highest radioactivity concentration at 30 min is found in the cerebellum (SUV 2.28), followed by the cortex. For all brain regions the radioactivity concentration at 60 min p.i. is lower compared to 30 min time point, indicating that wash-out has started.
• Table 9 shows the % ID values at 2 min, 30 min and 60 min p.i. of the radiotracer.
• the total initial brain uptake of the tracer was 0.64 % of the ID, with 0.46 % ID in the cerebrum and 0.15 % ID in the cerebellum.
• At 2 min p.i.6.0 % of the ID was present in the blood, and this cleared to 3.4 % by 60 min p.i.
• the tracer was cleared mainly by the hepatobiliary system as there was in total 25.5 % of ID present in liver and intestines 60 min after injection of the radiotracer.
• Hippocampus 0.017 ⁇ 0.005 0.021 ⁇ 0.004 0.026 ⁇ 0.002
• % ID/g tissue values were normalized for body weight.
• the normalized values for striatum, hippocampus, cortex and cerebellum are presented in Table 10.
• the radioactivity concentration has increased for almost all brain regions (small decrease for hippocampus but this can be due to an unpunctual dissection of this small brain region). This accumulation of radioactivity in these brain regions is consistent with the fact that mGluR2 receptors are expressed in several brain areas including hippocampus, cortical regions, olfactory bulb, cerebellum and striatum. Most significant increase was observed for cortex (SUV 1.16 at 2 min p.i. to SUV 1.39 at 30 min p.i.). The highest radioactivity concentration at 30 min is found in the cerebellum (SUV 1.68).
• Table 11 shows the % ID values at 2 min, 30 min and 60 min p.i. of the radiotracer. At 2 min p.i. 8.5 % of the ID was present in the blood, and this cleared to 2.9 % by 60 min after injection of the tracer. The total initial brain uptake of the tracer was 0.75 %, with 0.54 % of the ID in the cerebrum and 0.17 % in the cerebellum. At 60 min after injection of the radiotracer, 38.4 % ID was present in the liver and intestines.
• [ u C]B-2 has the highest total brain uptake at 2 and 30 min p.i. From these biodistribution studies, [ u C]B-2 looks the most promising PET tracer for in vivo mGluR2 imaging. Table 13. Comparative total brain uptake in normal rats at 2, 30 and 60 min p.i. for all six studied u C-labelled tracers
• [ u C]B-2 is most stable in plasma with 70 % of the recovered radioactivity present as the intact tracer 30 min p.i. Table 14. Relative percentages of intact tracer and radiometabolites in rat plasma at 30 minp.i. of[ n C]B-2, [ n C]B-4, [ n C]B-7, and[ n C]B-10
• the relative amounts of parent tracer and radiometabolites in perfused cerebellum and cerebrum at 30 min p.i. of the tracer was determined in healthy male Wistar rats for [ u C]B-4, [ n C]B-2, [ u C]B-7, and [ U C]B-10.
• rats were sacrificed by administering an overdose of Nembutal (CEVA Sante Animale, 200 mg/kg intraperitoneal).
• Nembutal CEVA Sante Animale, 200 mg/kg intraperitoneal
• the cerebrum/cerebellum extract was then injected onto an HPLC system consisting of an analytical XB ridge® column (Cis, 5 ⁇ , 3 mm x 100 mm, Waters) eluted with a mixture of 0.05 M NaOAc buffer (pH 5.5) and CH 3 CN (60:40 v/v) at a flow rate of 0.8 mL/min.
• the HPLC eluate was collected as 1 mL fractions (fraction collection each minute) after passing through the UV detector (254 nm), and the radioactivity in the fractions was measured using an automated gamma counter.
• Imaging experiments were performed on a Focus TM 220 microPET scanner (Concorde Microsystems, Knoxville, TN, USA) using healthy male Wistar rats. During all scan sessions, animals were kept under gas anesthesia (2.5 % isoflurane in 0 2 at 1 L/min flow rate).
• Dynamic scans of 90 min were acquired. After reconstruction of the images (filtered back projection), they were spatially normalized to an in- house created [ u C]raclopride template of the rat brain in Paxinos coordinates. Automated and symmetric volumes of interest (VOIs) were generated for different brain regions (striatum, cortex, cerebellum, hippocampus, hypothalamus, thalamus, substantia nigra, nucleus accumbens and lateral globus pallidus) from which time-activity curves (TAC) were constructed for each individual scan, using PMOD software (v 3.1, PMOD Technologies Ltd.). The radioactivity concentration in the different brain regions was expressed as SUV as a function of time p.i. of the radiotracer by normalization for body weight of the animal and injected dose.
• VOIs Automated and symmetric volumes of interest
• Rats were injected with 30-60 MBq of high specific activity formulation of [ u C]B-4, [ u C]B-2, [ n C]B-7, or [ n C]B-10 via the tail vein under isoflurane anesthesia (2.5 % in 0 2 at 1 L/min flow rate).
• compound A compound B or ritanserin were dissolved and administered in a vehicle containing 20 % (2- hydroxypropyl)-P-cyclodextrine and two equivalents hydrochloric acid.
• the ritanserin solution was protected from light.
• Compound A and compound B have affinity for mGluR2.
• a self-blocking study was done by subcutaneous (s.c.) administration of the authentic reference material (for [ u C]B-4) at ⁇ 30 min prior to the radiotracer injection.
• Displacement studies were performed by i.v. injection of compound B at dose 4, 1, 0.3 and 0.1 mg/kg, compound A at dose 1 mg/kg or ritanserin at dose 0.3 mg/kg. All chase compounds were injected -30 min after radiotracer injection. A wash-out period of at least four days was maintained between the different pretreatment and displacement studies.
• [ u C]B-4 was evaluated in vivo in three rats which were scanned dynamically for 90 min using ⁇ .
• the first rat was used for a baseline scan.
• the second rat was pretreated with authentic reference material B-4 via s.c. administration (dose 10 mg/kg) at 30 min prior to tracer injection.
• the third rat was used in a chase experiment and was injected i.v. with authentic reference material B-4 (dose 3 mg/kg) 30 min after tracer injection.
• a chase experiment was performed for [ u C]B-2 with different doses of compound B (4, 1, 0.3, 0.1 mg/kg).
• the chase compound was injected i.v. 30 min after tracer injection.
• Table 17 gives an overview of the average SUV values before and after injection of the chase for the total brain. This study shows that there is a clear relationship between the administered dose of the chase compound B and the receptor occupancy.
• SUV values are averaged values. Before chase injection: averaged values of time period 930- 1650 sec p.i. After chase injection: averaged values of time period 4650-5250 sec p.i.)
• additional chase experiments were performed with compound A, an compound with high selectivity for mGluR2.
• an additional chase experiment was performed with ritanserin, a 5HT 2 antagonist.

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